Autoimmune diseases represent a major health concern. For example, type 1 diabetes (T1D) is an autoimmune disease mediated by autoreactive T cells that recognize β-cell antigens (Ags), leading to destruction of pancreatic islets. A major problem in T1D management is its late diagnosis. This typically takes place after a variable period of subclinical, silent autoimmunity, once a significant proportion of β cells have already been destroyed. The resulting insulin deficiency leads to hyperglycemia and clinical onset. Hence, T1D prevention and treatment should target the underlying autoimmune mechanisms rather than its metabolic consequences, as done today with insulin replacement therapies. However, immunotherapies aimed at blunting β-cell autoimmunity need to have an excellent safety profile, since T1D mostly affects children and young adults and is not life-threatening in the short term.
β-cell Ag-specific therapies are therefore attractive in light of their selectivity and safety, compared to treatments broadly targeting the T-cell subsets involved in disease1. Such therapies are administered in the form of vaccines comprising the β-cell Ags whose immune recognition mediates islet destruction. These vaccines are formulated to achieve immune outcomes that are opposite to those pursued with classical vaccination, i.e. to neutralize rather than to stimulate the T-cell responses against the administered Ags—instating a condition known as immune tolerance. Clinical trials have however been deceiving2-4. Several attempts have focused on tolerogenic vaccination with β-cell Ags derived from insulin and its precursor preproinsulin (PPI), since this is the initiating Ag in the non-obese diabetic (NOD)5,6 mouse and likely also in humans2. A recent trial employing intranasal insulin administration to halt autoimmune β-cell destruction in new-onset T1D patients with slowly evolving disease did not result in significant β-cell preservation, despite evidence that insulin-specific immune tolerance was successfully induced7. These results suggest that we may need to intervene earlier, before significant β-cell loss and before autoimmune progression. While PPI recognition initiates the autoimmune cascade, subsequent β-cell destruction releases additional Ags that are further recognized (a phenomenon known as Ag spreading), thus making tolerance restoration vis-à-vis of the sole PPI insufficient.
The same problem is encountered in prevention trials using insulin vaccination, where the safety issue is even more critical for treating at-risk subjects who are not yet diabetic. Despite absence of clinical disease, selection of at-risk subjects is based on positivity for multiple auto-antibodies (auto-Abs), which witness an autoimmune reaction that already involves several Ags3, 8, 9. Recent studies further suggest that β-cell autoimmunity initiates very early, as the median age at auto-Ab seroconversion was only 9-18 months in large prospective cohorts of genetically at-risk children10,11. Insulin Ag-specific prevention strategies should therefore be implemented at a much earlier stage, in at-risk children (i.e. first-degree relatives of T1D patients) carrying a high HLA-associated genetic risk of disease but with no signs of active autoimmunity (i.e. auto-Ab-)12,13.
The perinatal period offers such opportunities not only in terms of timing, but also because it is characterized by immune responses to introduced Ags that are biased towards tolerogenic outcomes. Indeed, Ag introduction during fetal and neonatal life results in Ag-specific immune tolerance persisting during adulthood14-16. A key role in this process is played by central tolerance, a process taking place in the thymus in which developing T cells are challenged with ectopically expressed self-tissue Ags such as PPI. Their recognition leads to elimination of autoreactive pathogenic T effector cells (Teffs) and to positive selection of T regulatory cells (Tregs)17. This process is very active during the perinatal period and leads to the definition of immunological self that later imprints peripheral immune responses. The ‘immune self-image’ presented in the thymus is however incomplete, because the self Ag repertoire is only partially expressed17. Indeed, defective central tolerance is the first checkpoint in T1D progression. Some autoreactive Teffs escape thymic selection and can later be activated in the periphery and perpetrate islet destruction. Supporting this notion, the NOD mouse model of T1D develops accelerated diabetes when PPI expression is abolished in the thymus (Ins2−/− NOD mice)18,19. Second, human INS polymorphic variants predispose to T1D by decreasing INS expression in the thymus20.
However, this knowledge has not translated into therapeutic strategies aimed at boosting central tolerance ab initio. All tolerogenic vaccination approaches explored to date using the subcutaneous, intranasal or oral route are targeted on peripheral tolerance and aim at blunting the pathogenic potential of autoreactive Teffs and/or at enhancing Treg activity1. If we could instead introduce self Ags such as PPI in the thymus during the perinatal period, we could boost the T-cell selection process and intervene on the very first step in autoimmune progression.
The present invention relates to the use of Ags fused with the Fc portion of an IgG to induce immune tolerance by mucosal vaccination. Fc-fusion proteins currently represent 20% of all Ab-based medicines with FDA approval and are actively investigated in a variety of settings because addition of an Fc moiety increases the half-life of protein therapeutics. Binding of the Fc domain to the neonatal Fc receptor (FcRn) expressed in endothelial cells leads to their transient intracellular sequestration and slow release in the circulation. The FcRn is a heterodimer composed of a major histocompatibility complex (MHC) Class-I-like heavy chain and β2-microglobulin21. The interaction between IgG and FcRn requires an acidic pH (<6.5) and is inefficient at a physiological pH (7.4)21. It occurs in a 1:2 stoichiometry, with one IgG binding to two FcRn molecules via the FcRn heavy chains and the CH2-CH3 portion of the Fc domain of IgG22.
Besides endothelial cells, the FcRn is expressed in several other tissues and cells23, including the placental syncytiotrophoblast or yolk sac of mammals, the liver, intestinal, bronchial, renal (proximal convoluted tubule), genital, ocular and choroid plexus epithelia, renal podocytes; the skin (hair follicles, sebaceous glands, epidermal keratinocytes and melanocytes); hematopoietic cells such as dendritic cells, monocytes and macrophages (including macrophages in the lamina propria of the small intestine)24. In the respiratory tract, the FcRn is predominantly found in the bronchial epithelium of upper and central airways. In the human digestive tract, FcRn is expressed in epithelial cells of the stomach, the small intestine (duodenum, jejunum and ileum) and the colon25-27. There is an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression in the intestinal tract, with expression being the lowest in the duodenum-jejunum and highest in the proximal colon. This expression gradient correlates with the efficiency of in vitro monoclonal Ab (mAb) transcytosis for these different intestinal regions, with systemic entry occurring via the lymphatics27. The same expression gradient is found in cynomolgus monkeys, in which serum mAb levels were greater after ileum-proximal colon infusion than after administration into the duodenum-jejunum27. Taken together, the FcRn expression and mAb uptake patterns suggest that the ileum-proximal colon is the region where most of the mAb is transcytosed through the FcRn.
In more recent years, the application of Fc-coupled agents has therefore been extended to strategies aimed at delivering bioactive molecules using less invasive administration route, namely the gastrointestinal or pulmonary route. Typically, IgG are endocytosed on the apical membrane of epithelial cells and bind to FcRn at the acidic pH present in endosomes. The vesicle then fuses again with the basolateral membrane, where the extracellular neutral pH promotes the dissociation of IgG from FcRn. Importantly, FcRn-mediated transport of IgG is bidirectional (i.e. both from the apical and basolateral membrane of epithelial cells)28 and occurs rapidly, within 1 h after IgG addition in in vitro Transwell experiments26. Examples of Fc-coupled agents explored for oral delivery include Fc-coupled follicle-stimulating hormone (FSH)29, IgG mAbs27 and Fc-coated nanoparticles containing bioactive molecules such as insulin30. Overall, the systemic bioavailability achieved by oral administration of Fc-coupled agents is relatively limited. Examples of Fc-coupled agents explored for pulmonary delivery include Fc-coupled erythropoietin31-33 and Fc-coupled FSH29.
Hence, Fc-coupled agents have been used to increase the half-life of systemically administered therapeutic proteins and to facilitate their systemic delivery through the intestinal or bronchial route. We here propose to use the same strategy to induce immune tolerance. To this end, Ags can be modified by fusing them to the Fc portion of an IgG1, thus allowing the resulting Ag-Fc proteins to interact with the FcRn and cross mucosal barriers. This is the same pathway that physiologically delivers maternal IgG to foetuses (through the placenta) and to newborns (through the gut, during lactation)21, thus providing passive IgG protection during the intrauterine period and the first 6 months of life, when IgG production is not yet operational. This concept was first validated using the transplacental route of transfer and indicated that Fc-fused Ags intravenously administered to pregnant mice reach the fetal thymus in an FcRn-dependent manner and promote the generation of Ag-specific Tregs, leading to Ag-specific tolerance24. When applied to T1D mouse models, PPI-Fc transplacental transfer protects the offspring from diabetes development later in life35,36. A single 100 μg PPI-Fc dose intravenously administered to pregnant PPI T-cell receptor-transgenic G9C8 NOD mice (in which all T cells recognize a PPIB15-23 epitope37) is transplacentally transferred and protects the offspring from diabetes, without inducing metabolic or other adverse effects. This transfer is Fc- and FcRn-dependent. Diabetes protection is associated with peripheral PPI-reactive CD8+ Teffs displaying impaired cytotoxicity and with increased thymic-derived neuropilin-1 (NRP1/CD304)+ CD4+ Tregs secreting the regulatory cytokine transforming growth factor (TGF)-β. PPI-Fc reaches the thymus carried by migratory (CD8loCD11b+SIRPα+) dendritic cells (DCs) and diabetes protection is lost when migration is inhibited by early administration of anti-vascular cell adhesion molecule (VCAM)-1 Abs. Importantly, diabetes protection is also active in polyclonal NOD mice35. Although successful, this strategy remains invasive for autoimmune diseases like T1D that we cannot predict with certainty early enough, a fortiori prenatally. Such strategies applied to pregnant women may be considered to carry a risk for the fetus and the mother unacceptable in front of diseases that are in most cases not life-threatening in the short term and that may or may not develop.